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Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy
Joanna Xiuzhu Xu1, Md Siddik Alom1, Nicholas C Fitzkee1
1Department of Chemistry, Mississippi State University, Starkville, Mississippi 39762, United States.
Analytical Chemistry
|August 25, 2021
Summary
A new referencing system using 15N tryptophan (Trp) enables precise quantification of protein adsorption onto nanoparticles (NPs) using nuclear magnetic resonance (NMR). This method accurately measures competitive binding kinetics and thermodynamics in complex protein mixtures.
Area of Science:
- Biophysical Chemistry
- Materials Science
- Biochemistry
Background:
- Quantitative nuclear magnetic resonance (NMR) relies on effective intensity-based references for accurate molecular concentration measurements.
- Existing NMR referencing systems are not optimized for complex multidimensional experiments, particularly for studying protein-nanoparticle (NP) interactions.
- Understanding competitive protein adsorption onto NP surfaces is crucial in various biological and biomedical applications.
Purpose of the Study:
- To develop and validate a novel, simple external referencing system for quantitative 1H-15N 2D NMR experiments.
- To enable precise measurement of competitive protein adsorption onto NP surfaces, including binding kinetics and thermodynamics.
- To accurately quantify protein signals in mixtures and monitor in situ interactions.
Main Methods:
- Development of a referencing system using 15N-labeled tryptophan (Trp) as an external standard.
- Validation using gold nanoparticles (NPs) and single protein adsorption measurements.
- Application to protein mixtures, including variants differing by a single residue, using 1H-15N 2D NMR.
Main Results:
- The Trp referencing system accurately calibrates residue peak intensities and reduces systematic errors in NMR quantification.
- Precise determination of single protein binding capacity onto gold NPs was achieved.
- Accurate quantification of signals from individual proteins in mixtures and real-time monitoring of competitive adsorption kinetics were demonstrated.
- The study successfully analyzed the competitive binding of protein variants differing by a single residue.
Conclusions:
- The developed 15N Trp referencing system significantly enhances the precision and accuracy of quantitative NMR studies of protein-NP interactions.
- This method enables in situ and real-time kinetic monitoring of competitive protein adsorption, providing insights into NP corona formation.
- The referencing system is broadly applicable to various 1H-15N 2D NMR techniques for studying complex biomolecular interactions.
